Process for the production of beta-carotene



United States Patent 3,242,054 PROCESS FOR THE PRODUCTION OF p-CAROTENELeon Ninet and Jacques Albert Renaut, Paris, and Robert Charles FrancoisTissier, Maisons-Alfort, France, assignors t0 Rhone-Pouleuc S.A., Paris,France, a corporation of France No Drawing. Filed Mar. 4, 1963, Ser. No.262,369 5 Claims. (Cl. 195-28) The present invention relates to animproved process for the production of fi-carotene by fermentation.

It is known to obtain fi-carotene by submerged fermentation ofmicroorganisms of the genus Choanephora or Blakeslea. Variouspublications have described the conditions which favour the productionof fi-carotene. Barnett and his co-workers (Science, 123, 141 (1956))have shown that the production of B-carotene is improved by thesimultaneous culture of the opposite forms of one species. This studywas thereafter extended to the culture of opposite forms of diiferentspecies (C. Hesseltine, Mycologia, 49, 449 (1957)) The culture mediawere also examined and it was found that the addition of whole orhydrolysed seeds, of vegetable oils, of surface-active agents, ofantioxidants and of thickening agents increased the fi-carotene yield.(R. Anderson and others, J. Agr. Food Chem. 6, 543 (1958).) (A. Cieglerand others, App. Microb. 7, 94 and 98 (1959).)

Finally, Mackinney and his co-workers (J. Am. Chem. Soc. 74, 3456(1952)) have shown that the addition of B-ionone to the static cultureof a Phycomyces greatly increases the formation of B-carotene to thedetriment of other carotenoid pigments. Anderson and others noted thesame effect in agitated culture with Blakeslea and Choanephora (loc.cit.),

It has now been found, and this forms the subject of the presentinvention, that it is advantageous to replace 5- ionone in culture mediaby 2,2,6-trimethylcyclohexanone or its functional derivatives. Suchcompounds are less complex and therefore more readily obtainable than 5-ionone, and their use results in a considerable increase in the rate ofproduction of fi carotene as compared with reference media containing noprecursor.

By functional derivatives are meant the products of condensation of2,2,6-trimethylcyclohexanone with ammonia or its monosubstitutedderivatives, such as the oxime, the hydrazone, the semicarbazone, thethiosemicarbazone or the imines, all of which, in common with 2,2,6-trimethylcyclohexanone itself, are 2,2,6-trimethylcyclohexanederivatives double-bonded at the l-carbon atom to a functional atom orgroup. Of these derivatives of 2,2,6-trimethylcyclohexanone, thesemicarbazone is preferred.

2,2,6-trimethylcyclohexanone (or its derivative) may be added to theculture medium in quantities ranging from 0.1 to g./litre, at thecommencement or during the course of the fermentation, in one or morelots. Preferably, a quantity between 0.8 and 3 g./litre is employed andthe addition is carried out after 1 to 3 days of culture. Regardless ofthe quantity of 2,2,6-trirnethylcyclohexanone or its derivative which isadded, and regardless of the time of the addition, it is desirable tocontinue the culture for 6 to 10 days after the inoculation in order toobtain maximum production of B-carotene. The culture medium may vary,but contains essentially a source of assimilable carbon and a source ofassimilable nitrogen, mineral elements and, where necessary, growthfactors, antioxidants, surface-active agents and thickeners.

A source of assimilable carbon there may be used carbohydrates (such asglucose, dextrins and starch), animal or vegetable oils (such as lardoil, soya bean oil,

3,242,054 Patented Mar. 22, 1966 and cotton seed oil) or mixturesthereof. The suitable sources of assimilable nitrogen are extremelyvaried, and they may be defined chemical compounds or complex substancescontaining nitrogen mainly in protein form, such as casein, lactalbumin,gluten and their hydrolysates, soya bean fluor, peanut fluor, yeastextracts, distillers solubles and corn-steep.

Some of the mineral elements added may have a buffer or neutralisingeffect, such as alkali or alkaline-earth phosphates.

The most frequently employed of the growth factors is vitamin B orthiamine. Of the antioxidants, there may be mentionedN,N'-diphenylparaphenylenediamine, 2,2,4-trimethyl-6-ethoxy-1,2-dihydroquinoline, ascorbic acid or sorbic acid.The surface-active agents are preferably of the non-ionic type, such asderivatives of sorbitol with fatty acids, or products based on ethyleneoxide condensation products. The most commonly employed thickeners arestarch, carboxymethylcellulose and agar.

It has been found that in these media 2,2,6-trimethylcyclohexanone andits derivatives are compatible with the auxiliary substances describedin the French patent application No. 899,751, filed on June 5, 1962,.under the title Process for the Production of B-Carotene, that is tosay: with amides of the general formula:

RCONR R (in which R represents a hydrogen atom, an alkyl radical havingat most 6 carbon atoms or an aryl or aralkyl radical, and R and R whichare identical or different, each represent a hydrogen atom or an alkylradical having at most 4 carbon atoms, or dialkylsulphoxides whose alkylradicals contain at most 4 carbon atoms. The culture medium thusprepared is thereafter inoculated with a culture of the and forms ofBlakeslea trispora (NRRL 2456 and 2457).

The following examples illustrate the invention:

Example I A number of 300 cc. Erlenmeyer flasks are filled with 50 cc.of a culture medium having the following composition:

Corn seed hydrolysate g Soya bean oil cc- 20 Cotton seed oil cc 20 Tweencc 1.2 Potassium monophosphate g 0.5 Carboxymethylcellulose g 20Thiamine hydrochloride g 0.01

Distilled water to make 1000 cc.

The pH of the medium is adjusted to 61.2 with concentrated sodiumhydroxide. The flasks are sterilised for 20 minutes at C. After cooling,there is added under sterile conditions to each flask 0.5 cc. of asterile 1% 1,2- dihydro-2,2,4-trimethyl-6-ethoxyquinoline solution inpetroleum.

Each flask is thereafter inoculated with 5 cc. of an agitated culturecontaining the two forms, and of the Blakeslea trispora strain (NRRL2456 and NRRL 2457), and aged 24 hours.

The flasks are thereafter placed on a rotary agitating table turning at220 r.p.m. in a chamber at 27 C. After incubation for 2 days, thecontents of the flasks are divided into two equal lots.

To each flask of the first lot is added under sterile conditions 0.5 cc.of petroleum.

Each flask of the second lot receives 0.5 cc. of a 10%2,2,6-trlmethylcyclohexanone solution in petroleum.

The cultures are continued under the same conditions of temperature andagitation for 6 further days. At the .4. end of this time, theproduction of fl-carotene is maximum in all the flasks.

The yield of ,B-carotene is determined as follows: the mycelium isseparated by filtration, washed with Water and then dried overnight invacuo at 35 C. The dry mycelium is thereafter extracted by means ofhexane. The ti-carotene is separated from the other carotenoids presentby chromatography of the extract through alumina. The elution fractionscontaining fi-carotene are combined and spectrophotometrically titratedas compared With a pure specimen of ,B-carotene.

The productions are as follows:

2,2,6'trimethylcyclohexanone, ,B-Carotene,

g./l. of initial medium: mg./l. of broth 0 105 1 170 Example 11 500 cc.of Water containing 75 g. of distillers solubles are boiled for minutes.After cooling, there are added:

Corn starch g 50 Soya bean oil cc 30 Cotton seed oil cc 30 Tween 80 cc1.2 Yeast extract g 1 Potassium monophosphate g 0.5N,N'-dipheny1paraphenylenediamine g 0.1 Thiamine hydrochloride g 0.01

The volume is made up to 1000 cc. with distilled water.

The mixture is adjusted to a pH of 6.0 with sodium hydroxide,distributed in 300 cc. Erlenmeyer flasks (50 cc. per flask) and thensterilised for minutes at 120 C. After cooling, 0.5 cc. of petroleum isadded to each flask under sterile conditions.

The inoculation with a mixed culture of the and forms of Blakesleazrispora and the development of the inoculated cultures take place underthe conditions described in Example I.

After development for 48 hours, the culture flasks are distributed in 4series and each flask receives the following additions under sterileconditions:

1st series: petroleum 0.5 cc.

2nd series: 50 mg. of 2,2,6-trimethylcyclohexanone in solution in 0.5cc. of petroleum.

3rd series: 100 mg. of 2,2,6-trimethylcyclohexanone in solution in 0.5cc. of petroleum.

4th series: 150 mg. of 2,2,6-trimethylcyclohexanone in solution in 0.5cc. of petroleum.

The cultures are continued for a further 8 days. The ,8carotene yields,determined as in Example I, are as follows:

,B-Carotene, mg./1. of broth 2,2,6-trimethylcyclohexanone,

g./ litre of medium:

Example 111 500 cc. of water containing 75 g. of distillers solubles areboiled for 15 minutes. After cooling, there are The volume is made up to1000 cc. with distilled water. The mixture is adjusted to a pH of 6.0with sodium hydroxide and distributed in 300 cc. Erlenmeyer flasks (50cc. per flask). After sterilisation for 20 minutes at 120 C. andcooling, there is added to each flask under sterile conditions 0.5 cc.of a 1% 2,2,4-trimet-hyl-6-ethoxy-l,2-dihyd-roquinoline solution inpetroleum.

Inoculation with a mixed culture of the and forms of Blakeslea tris oraand development of the inoculated cultures takes place under theconditions indicated in Example I.

After development for 48 hours, the culture flasks are distributed in 3series and each Erlenmeyer flask receives the following additions understerile conditions:

1st series: 0.5 cc. of petroleum.

2nd series: 50 mg. of 2,2,6-trimethylcyclohexanone in solution in 0.5cc. of petroleum.

3rd series: 60 mg. of semicanbazone of 2,2,6-trirnethylcy- 'clohexanonein the form of a 7% suspension in water containing 1% of Tween and 0.5cc. of petroleum.

After these additions, the cultures are continued [for 6 more days underthe same conditions of temperature and agitation. After this time, the{i-carotene production is maximum in all the flasks.

The yields of B-carotene, determined as in Example I, are as follows:

Addition at 48 hours: Yield of B-carotene in mg./l. of broth Petroleum820 2,2,6-trimethylcyclohexanone+pet1'oleum 940 Semicarbazone of2,2,6-trimethylcyclohexanone-l-petroleu-m 1110 Example IV Into a 30litre fermentation vessel are introduced: Distillers solubles g 1500Water litres 11 The mixture is stirred and heated at C. for d0 minutes.After cooling, the pH is adjusted to 6.4 with 70 cc. of 10 N sodiumhydroxide and the medium is made up with the following startingmaterials:

Starch g 750 Soyabean oil cc 450 Cotton seed oil cc 450 Tween 80 c c 75Yeast extract g 15 Potassium monophosphate 'g 7.5 Manganese sulphatemonohydrate g 1.5 2,2,4-trimethyl-6-ethoxy-1,2-dihydroquinoline cc 1.5Thiamine hydrochloride solution (0.3 g./

litre) cc 250 The volume is adjusted to 15 litres with water, the pH ofthe mixture being at 6.40. The medium is sterilized for 50 minutes at122 C. With injection of steam. After cooling, the pH is 5.85 and thevolume is 14.2 litres.

There are then added under sterile conditions 30 cc. ofdimethylformamide and cc. of petroleum. The fermentation vessel isthereafter inoculated with two inoculum cultures in fermentationvessels, aged 48 hours, of the strain Blakeslea trispora in a portion of750 cc. of culture of the form (NRRL 2456) and 750 cc. of culture of theform NRRL 2457). The culture is developed at 26 27 C. while stirring bymeans of a turbine at 400 rpm. and aerating with a rate of flow of 1.2m. /h.

After development for 48 hours, 15 cc. of 2,2,6-trimethylcyclohexanonein solution in 150 cc. of petroleum are added under sterile conditions.After this addition, the culture is continued under the same conditionsof aera tion, agitation and temperature for 5 further days.

The production of ,B-carotene, titrated as indicated in Example I, thenreaches 580 rug/1.

In another 30 litre fermentation vessel, the above'described medium isprepared in the same way. After sterilisation and cooling, there arealso added 30 cc. of dimethylformamide and 150 cc. of petroleum. Themedium is thereafter inoculated under the conditions described in theforegoing with the same inoculum cultures. After development for 48hours, 150 cc. of petroleum and 21 g. of 2,2,6-trirnethylcyclohexanonesemicar-bazone in the form of a 7% suspension in water containing 1% OctTween 80 are added under sterile conditions. After these additions, theculture is continued under the same conditions of aeration, agitationand temperature for 5 further days.

The fl-carotene production is then 765 mg./ 1.

We claim:

1. Process tfOX the production of li-car-otene which comprises the stepsof (a) inoculating with a mixture of the l and forms of Blakesleatrispora a medium containing a source of assimila ble nitrogen, a sourceof assimila'ble carbon, mineral elements, and2,2,6-trirnethy1cyclohexanone or 2,2,6-trimethylcyclohexanonesemicarbazone, to form a culture, (b) fermenting the culture underaerobic conditions, and (c) recovering {i-carotene 'from the culture.

2. Process according to claim 1 wherein the 2,2,6-trimethylcyclohexanoneor 2,2,6-trimethylcyclohexanone semicar bazone is present in the mediumin an amount of up to 3 g. per litre of medium.

3. Process .for the production of ,B-carotene which comprises the stepsof (a) inoculating with a mixture of the and forms of Blakeslea trisporaa medium containing a source of assimilable nitrogen, a source ofassimilalble carbon, and mineral elements to form a culture, (b)fermenting the culture under aerobic conditions, adding to the culture2,2,6-trimethylcyclohexanone or 2,2,6-trimethylcyclohexanonesemicanbazone, (d) continuing to ferment the culture under aerobicconditions, and (e) recovering fi-carotene trom the culture.

4. Process according to claim 3 wherein the 2,2,6-trimethylcyclohexanone or 2,2,6-trimethylcyclohexanone semi'car bazone is addedto the culture after fermentation has proceeded for 1 to 3 days, andfermentation is thereafter continued until 6 to 10 days after theinoculation.

5. Process for the production of fi-carotene which comprises the stepsof (a) inoculating vvtih a mixture of the and forms of Blakesleatrispora a medium containing a source otf assimilable nitrogen, a sourceof assimilable carbon, and mineral elements to form a culture, b)fermenting t-he culture under aerobic conditions for 1 to 3 days, (c)adding to the culture 2,2,6-trirnethylcyclohexanone or2,2,6-tri-methylcyclohexanone semicar-bazone in an amount of up to 3 g.per litre of culture, (d) continuing to fenrnent the culture underaerobic conditions until 6 to 10 days after the inoculation, and (e)recovering fl-carotene from the culture.

Organic Chemistry, by Karrer, 2nd ed., Elsevier Publishing Co, Inc., NewYork, 1946, pp. 666 to 672.

A. LOU-IS MONACELL, Primary Examiner.

1. PROCESS FOR THE PRODUCTION OF B-CAROTENE WHICH COMPRISES THE STEPS OF(A) INOCULATING WITH A MIXTURE OF THE + AND - FORMS OF BLAKESLEATRISPORA A MEDIUM CONTAINING A SOURCE OF ASSIMILABLE NITROGEN, A SOURCEOF ASSIMILABLE CARBON, MINERAL ELEMENTS, AND2,2,6-TRIMETHYLCYCLOHEXANONE OR 2,2,6-TRIMETHYLCYCLOHEXANONESEMICARBAZONE, TO FORM A CULTURE, (B) FERMENTING THE CULTURE UNDERAEROBIC CONDITIONS, AND (C) RECOVERING B-CAROTENE FROM THE CULTURE.